The Lichenologist 45(6): 841–856 (2013) 6 British Society, 2013 doi:10.1017/S002428291300056X

Cetraria steppae Savicz is conspecific with (Schreb.) Fr. according to morphology, secondary chemistry and ecology

Olga NADYEINA, Tetiana LUTSAK, Oleg BLUM, Volodymyr GRAKHOV and Christoph SCHEIDEGGER

Abstract: Eurasian Cetraria steppae and the more widely distributed C. aculeata are two lichen species traditionally distinguished by Eastern European and Spanish lichenologists on the basis of their morphological and ecological characteristics. Other specialists, however, consider them puzzling. This paper aims to evaluate the taxonomic status of these members of the C. aculeata group and thereby to clarify their conservation status in Ukraine. Morphological, chemical and ecological features of specimens originating from populations in different regions of Ukraine were tested and compared with the main characteristics commonly used for the species delimitation. Neither morphological nor chemical traits were found to correlate with ecological characteristics on a small geographical scale. Variation in the norstictic acid content detected in 256 individuals from 13 populations in Ukraine showed no correlation with the morphological characteristics that are currently used for species de- limitation. These morphological features appear to vary continuously and did not support subdivision among the specimens studied. We hypothesize that C. steppae and C. aculeata are conspecific, and provide a formal synonymy. Specimens with norstictic acid are regarded as a different chemotype. Possible evolutionary and adaptive roles of norstictic acid in C. aculeata s. lat. are discussed. Based on current and historical data, we consider C. aculeata s. lat. as vulnerable in Ukraine, according to the IUCN criteria for regional Red List assessment. Key words: IUCN, , norstictic acid, populations, Red Data Book, Red List, Ukraine Accepted for publication 4 July 2013

Introduction australiensis W. A. Weber ex Ka¨rnefelt, C. crespoae (Barreno & Va´zquez) Ka¨rnefelt, C. Members of the Cetraria aculeata group muricata (Ach.) Eckfeldt, C. odontella (Ach.) are fruticose, brownish black lichens and Ach., ‘C. panamericana’andC. steppae (Savicz) they include C. aculeata (Schreb.) Fr., C. Ka¨rnefelt (Ka¨rnefelt et al. 1992; Thell et al. 2002; Printzen et al. 2013). Of these, C. aculeata and C. steppae have been considered O. Nadyeina: M. G. Kholodny Institute of Botany, Li- chenology and Bryology Department, Kyiv, Ukraine; and as two closely related species (Savicz 1924; Biodiversity and Conservation Biology, Swiss Federal In- Ka¨rnefelt 1986; Ka¨rnefelt et al. 1993). Cetra- stitute for Forest, Snow and Landscape Research (WSL), ria aculeata s. lat. was the subject of special Birmensdorf, Switzerland. Email: [email protected] taxonomic and phylogenetic studies (Ka¨rne- T. Lutsak: Taras Shevchenko National University of Kyiv, Department of Botany, Kyiv, Ukraine; and Senckenberg felt 1986; Ka¨rnefelt et al. 1992, 1993; Thell Research Institute and Natural History Museum, Frank- et al. 2000, 2002; Printzen et al. 2013). The furt on Main, Germany. phylogeography of its symbionts has been in- O. Blum: M. M. Gryshko National Botanical Garden of vestigated (Ferna´ndez-Mendoza et al. 2011; the Academy of Science of Ukraine, Bioindication and Domaschke et al. 2012; Printzen et al. 2013; Chemosystematics Laboratory, Kyiv, Ukraine. V. Grakhov: M. M. Gryshko National Botanical Garden Ferna´ndez-Mendoza & Printzen 2013), as of the Academy of Sciences of Ukraine, HPLC Center, well as the anatomy and ecophysiology of Kyiv, Ukraine. extreme morphotypes (Pe´rez-Ortega et al. C. Scheidegger: Biodiversity and Conservation Biology, 2012), and the bacterial communities within Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland. geographically distinct populations (Printzen 842 THE LICHENOLOGIST Vol. 45

Table 1. Features used for the delimitation of C. aculeata and C. steppae according to Savicz’s (1924) and Ka¨rnefelt’s (1986) descriptions.

Cetraria aculeata (Schreb.) Cetraria steppae (Savicz) Character Fr. Ka¨rnefelt Reference

Thallus vagrancy Attached tufts Different mode of life Savicz 1924 from C. aculeata Branches forming Branches forming Ka¨rnefelt 1986 eshrubby tufts, which espherical tufts com- arise directly from the posed of a few branches substratum; also tends due to the vagrant mode to develop spherical of life thalli, composed of more branches than thalli of C. steppae Thallus specific Glossy, solid, inflexible, Matt, smooth, soft and Savicz, 1924 features fragile flexible, not fragile Glossy to matt; black, brown to light brown; varying Ka¨rnefelt 1986 fragility Thallus size Not indicated Savicz 1924 2–4(–10) cm high 1–3 cm high Ka¨rnefelt 1986 Branch size Long and thin: Short and thick: Savicz 1924 0Á3–1Á0 mm wide 0Á4–2Á0 mm wide Main branches c.1(–4) Main branches c.0Á5– Ka¨rnefelt 1986 mm wide; terminal 2Á0(–4Á0) mm wide; branches c.0Á3–1Á0mm terminal branches wide c.0Á1–5Á0 mm wide Isidioid projections Tiny, rarely present Absent Savicz 1924 Absent or present Ka¨rnefelt 1986 Chemistry K-- K-- ,C-- ,I-- Savicz 1924 K-- ; norstictic acid K+; norstictic acid Ka¨rnefelt 1986 absent (TLC) present (TLC)

et al. 2012). However, the taxonomic status Mereschkowsky was the first to pay attention of C. steppae as a distinct species has been to vagrancy and the small number of branches questioned (Ferna´ndez-Mendoza et al. 2011; among specimens from Crimea (Ukraine). Lutsak et al. 2012) as its morphology varies His description is, however, very brief: ‘‘Thal- greatly and its distribution range overlaps lus libertus, opacus, laciniis paullulum minus with that of C. aculeata (Poelt 1969; Randlane attenuatis’’ (Mereschkowsky 1921). The au- & Saag 2006). thor of C. steppae at the species level is Savicz, Distinguishing Cetraria steppae and C. acu- who also gave a concise description (Table leata as distinct species was traditionally based 1). He emphasized that both taxa are distinct on visual morphological aspects (Savicz 1924; species without intermediate forms, which Oxner 1937; Poelt 1969), which include a implied that C. steppae occurs in the southern vagrant mode of life, the surface characteris- steppe biotopes and C. aculeata in the north tics of the thalli and morphological parameters (Savicz 1924). This statement guided lichen- of the main and terminal branches (Table 1). ologists to distinguish the two species, when 2013 Cetraria steppae is conspecific with C. aculeata—Nadyeina et al. 843 the vagrant mode of life or the thickness of ally used for the delimitation of C. steppae branches were not well developed. In the and C. aculeata, and to elucidate their taxo- 1980s, Ka¨rnefelt performed a wide-scale sur- nomic value. We also wanted to study the vey of the brown Cetraria species (Ka¨rnefelt impact of ecological and geographical factors 1986) and revised the traits for morphol- on the morphological and chemical charac- ogical distinction of the two species. He teristics within this group. We hypothesized introduced a new feature, the presence of that specimens belonging to C. steppae con- norstictic acid in C. steppae, which was not tain norstictic acid in the medulla (Ka¨rnefelt considered in the original description of C. 1986), have wider thallus branches, smaller steppae (Savicz 1924; Table 1) and became thalli, a vagrant lifestyle, and a distribution crucial for the delimitation of the two spe- limited to the south of Ukraine (Savicz 1924). cies. Moreover, Ka¨rnefelt extended the con- The aim was also to test the correlation be- cept of C. steppae and also included spe- tween the presence of norsticitic acid and cimens with isidioid projections and black, the content of carbonates in the substrata of glossy thalli (Table 1). After being described arid areas (Hauck et al. 2010). Finally, we by Savicz (1924), the species was reported assess and discuss the conservation status of in other parts of southern Ukraine (Oxner C. aculeata s. lat. according to the IUCN 1937, 1993; Blum 1996; Blum et al. 2009) Red List criteria for the next edition of the and Russia (Zhurbenko 1996; Moutchnik & Red Data Book of Ukraine. Zavarzin 2005; Golubkova et al. 2008; Urban- avichus 2010), Kazakhstan (Wagner & Spri- bille 2005), Turkey (John 1999; John & Materials and Methods Breuss 2004; Yazıcı et al. 2010), Iran (Soh- Materials and sampling design rabi & Alstrup 2007; Seaward et al. 2008), and Spain (Llimona & Hladun 2001). A total of 503 specimens of C. aculeata s. lat. from Ukraine were studied. First, we examined 247 speci- In our analysis of 247 herbarium speci- mens from different herbaria (KW, KHER, LE), ac- mens of C. aculeata s. lat., we were not able cording their morphological, chemical and ecological to classify all specimens as belonging to one characteristics (Fig. 1E). We then collected 256 speci- or other of the two taxa because of contradic- mens from 13 local populations, representing different tions or uncertainties in diagnostic traits natural zones of Ukraine during 2010–2011. Their char- acteristics are summarized in Table 2 and locations (Table 1). The features traditionally used to shown in Fig. 1E. All specimens were classified a priori distinguish them seemed to be quite subjec- as Cetraria aculeata s. lat. A tuft c.5–10cm2 was con- tive, such as the degree of vagrancy or the sidered to be one specimen (‘thallus’). To analyze the thickness of the branches. The vagrant life chemical variability among neighbouring thalli (which form and spherical tuft-like thalli are often are probably vegetative clones), we sampled thalli at 0Á5 m intervals along transects 10–25 m in length and 0Á5m difficult to designate, both on herbarium in width within each population. As a result, 5–41 speci- specimens and in the field. Specimens kept mens per population were collected according to their in herbaria are usually carefully separated, availability, amounting to 256 thalli in total. To study and could look ‘artificially vagrant’ (Fig. 1A the morphological variability within the local popula- tions, we selected a subset of 122 specimens, sampled at & C). In the field, the recognizable vagrant distances of >1 m along the same transects, resulting in thalli grow on bare soil or sand. However, a 5–18 specimens per population (Table 2). Because the few centimetres away, individuals may often species reproduces only asexually in Ukraine (no spe- be attached, which changes their degree of cimens with fruit bodies have been found so far in this area), we selected 1 m as the minimum distance to vagrancy (Fig. 1B & D). Delimitation of reduce the clones in our dataset because the range of individuals is also contentious because thalli dispersal through thallus fragments is considered to be in vagrant tufts or in mostly attached mats less than 1 m (Heinken 1999). are often densely entangled (Fahselt 2008). The above-mentioned difficulties motivated Morphological study us to check the morphological variability A subset of 122 air-dried specimens, collected from the within and between populations sampled 13 local populations mentioned above, was selected for from different regions in Ukraine. The pres- morphometric measurements (Tables 2 & 3). For each ent study aimed to test characters tradition- specimen, we measured the size of the whole thallus, 844 THE LICHENOLOGIST Vol. 45

Fig. 1. Morphological diversity and distribution of Cetraria aculeata s. lat. A, Crimea, Dzhankoj region, norstictic acid minor; B, Luhansk region, norstictic acid minor; C, type specimen (LE 220), Kherson region, norstictic acid major; D, Donetsk region, norstictic acid major; E, distribution in Ukraine, based on the analysis of 247 specimens (KW, KHER, LE) tested by TLC, numbers correspond to the sampling sites of the populations given in Table 2. Arrows indicate specimens selected for HPLC analysis (listed in Table 4); red circles, norstictic acid present; blue triangles, norstictic acid absent. Scales: A–D ¼ 1 cm. 2013 Cetraria steppae is conspecific with C. aculeata—Nadyeina et al. 845

Table 2. Populations of Cetraria aculeata s. lat. examined with characteristics of their sites (natural zones of Ukraine: F – polissia (forest), FS – forest-steppe, S –steppe, SSA – steppe semi-arid, SA – steppe arid, CM – Crimean Mountains; Habitats: O – open places, clearings among pine forest plantation, G – grasslands and heaths, Y – yayla or mountain top, deforested by grazing).

Number of specimens Total number selected for of specimens Natural morphometric analyzed Site Altitude, zone of study (including TLC, No. Population m a.s.l. Ukraine Habitat Substratum (n ¼ 122) n ¼ 256)

1 Shatsk* 170 F O Sand 11 41 2 Ratno 145 F O Sand 5 12 3 Platonivka 90 FS O Sand 9 16 4 Trehizbenka** 107 S G Sand 10 25 5 Stanychno-Luhansk** 55 S O Sand 10 20 6 Provallia** 170 S O Soil 10 21 7 Kamjany Mohyly† 160 S O Granite 14 22 8 Antratsit 140 S O Soil 6 15 9 Burkuty 15 SA G Sand 15 5 10 Kazantyp‡ 10 SSA G Limestone 11 23 11 Bondarenkovo 55 SSA G Soil 7 14 12 Arabat Split¶ 0 SA G Sand 6 11 13 Chatyr Dag 1340 CM Y Limestone 18 21

* Shatsk Lakes National Nature Park; ** Branch of Luhansk Steppe Nature Reserve; † Branch of Ukrainian Nature Steppe Reserve; ‡ Kazantyp Nature Reserve; ¶ Arabat Split Botanical Reserve. the width of the main branches at the basal bifurcations, with and without norstictic acid were present. Localities and the width of terminal branches below the last bifur- for specimens analyzed by HPLC are indicated in Fig. 1E cation (Table 3). Five measurements of each trait per with arrows and listed in Table 4. HPLC analyses were specimen were performed. The morphometric measure- carried out on the basis of methods described in Yoshi- ments are presented as (min–) mean eStandard Devia- mura et al. (1994), Gupta et al. (2007) and Manojlovic´ tion (–max). The presence or absence of isidioid projec- et al. (2010). The content of the lichen substances was tions was recorded for each specimen (Table 3). quantified according to a 10-point scale using peak heights at 224 nm and 1000 mAU detector signal range Chemical analyses for each specimen. A peak height between 500–1000 mAU was considered as ‘‘major’’ (5–10 points), 300– Each of the 247 herbarium specimens was tested by 500 mAU as ‘‘medium’’ (3–5 points), 30–300 mAU as spot reactions with 10% aqueous solution of potassium ‘‘minor’’ (1–3 points) and less than 30 mAU as ‘‘traces’’ hydroxide (K) and dissolved crystals of paraphenylene- (1 point). diamine in ethanol (Pd). The lichen substances were also analyzed using the microcrystal test method (Orange et al. 2001) for a subset of 17 selected specimens (Table 4). Statistical analyses Each of the 503 specimens studied were also analyzed by thin-layer chromatography (TLC), according to stan- Basic statistics for the morphometric parameters of the dard protocols (White & James 1985; Orange et al. C. aculeata s. lat. specimens collected from all 13 popu- 2001) using Sorbifil UV plates (Russia) and solvent A. lations, an analysis of variance (ANOVA) and the princi- High performance liquid chromatography (HPLC) pal component analysis (PCA) were run in JMP 9 (SAS was applied to a subset of 17 specimens, which were Institute Inc., Cary, North Carolina). The coefficient of selected according to the following criteria: 1) specimens variation (CV) was calculated for the entire dataset and originating from different parts of Ukraine (the Car- each population (Table 3). Values of CV were classified pathians, north-west, south-east and south Ukraine, in five categories: minor variation (0–10%), little varia- including Crimea); 2) individuals originating from local- tion (11–20%), average variation (21–40%), high varia- ities where, according to the TLC analysis, specimens tion (41–60%) and very high variation (61–100%). To 846

Table 3. Morphometric (n ¼ 122) and chemical (n ¼ 256) characteristics of the populations of C. aculeata s. lat. studied.

Width of the Width of the main branch (mm) terminal branch (mm) Thallus size (mm) Thalli with isidioid Thalli with norstictic e e e Site No. Population Mean SD CV, % Mean SD CV, % Mean SD CV, % projections, % acid, % H LICHENOLOGIST THE 1 Shatsk 1Á29 e 0Á16 12Á45 0Á47 e 0Á08 16Á00 3Á27 e 1Á26 38Á42 36 0 2 Ratno 1Á51 e 0Á28 18Á19 0Á40 e 0Á10 24Á85 6Á43 e 2Á00 31Á05 60 0 3 Platonivka 1Á44 e 0Á36 24Á63 0Á48 e 0Á06 12Á92 3Á57 e1Á06 29Á64 100 100 4 Trehizbenka 1Á15 e 0Á23 20Á37 0Á46 e 0Á06 13Á41 3Á20 e 0Á74 23Á23 30 100 5 Stanychno-Luhansk 1Á20e 0Á35 29Á40 0Á50 e 0Á10 20Á23 3Á21 e 0Á38 11Á96 20 90 6 Provallia 1Á57 e 0Á17 10Á81 0Á50 e 0Á03 6Á82 2Á26 e 0Á60 26Á50 10 80 7 Kamjany Mohyly 1Á62 e 0Á34 20Á84 0Á50 e 0Á03 6Á08 4Á33 e 1Á19 27Á43 43 64 8 Antratsit 1Á56 e 0Á16 10Á36 0Á50 e 0Á05 10Á00 2Á92 e 0Á26 8Á85 100 17 9 Burkuty 1Á25 e 0Á26 21Á13 0Á44 e 0Á08 18Á13 4Á47 e 1Á21 27Á00 40 100 10 Kazantyp 1Á32 e 0Á23 17Á76 0Á53 e 0Á06 11Á93 4Á19 e 1Á38 32Á92 64 73 11 Bondarenkovo 1Á41 e 0Á37 26Á34 0Á50 e 0Á02 4Á89 3Á40 e 0Á92 27Á04 29 100 12 Arabat Split 1Á75 e 0Á25 14Á42 0Á55 e 0Á06 10Á64 3Á05 e 0Á45 14Á77 0 100 13 Chatyr Dag 1Á28 e 0Á30 23Á56 0Á47 e 0Á06 12Á15 4Á45 e 0Á90 20Á18 100 22

Total in the dataset 1Á39 e 0Á32 22Á74 0Á49 e 0Á07 13Á81 3Á73 e 1Á31 34Á99 52 62 o.45 Vol. 2013 Cetraria steppae is conspecific with C. aculeata—Nadyeina et al. 847

Table 4. HPLC study of selected specimens of C. aculeata s. lat. from Ukraine

Herbarium No. Locality, collector, year TLC data* HPLC data**

LE220 (Type) Kherson reg., Chaplynsky distr. Oxner, 1923 NST, L NST (8), L (2), PL (1) KW5770 Kherson reg., Hola Pristan distr. Makarevych, 1950 NST, L, PL NST (4), L (4), PL (2) KW65049 Kherson reg., Bilozerka distr. Nadyeina, 2010 NST, L NST (3), L (5), PL (1) KW5824 Crimea, Dzhankoi reg. Osmanova, 1932 NST, L, PL NST (9), L (6), PL (2) KW66928 Luhansk reg., Sverdlovsk distr. Vasyluk, 2010 L, PL NST (2), L (3), PL (1) KW66930 Luhansk reg., Sverdlovsk distr. Vasyluk, 2010 NST, L NST (2), L (8) KW64472 Luhansk reg., Sverdlovsk distr. Nadyeina, 2005 L NST (1), L (6) KW64477 Luhansk reg., Sverdlovsk distr. Nadyeina, 2005 NST, L NST (2–3), L (3), PL (1) KW5855 Donetsk reg., Volodarsk distr. Oxner & L, PL NST (1), L (5), PL (1) Kopachevska, 1954 KW5856 Donetsk reg., Volodarsk distr. Oxner & NST, L NST (1), L (4), PL (1) Kopachevska, 1954 KW64475 Donetsk reg., Shakhtarsk distr. Nadyeina, 2006 L, PL NST (2), L (6) KW64476 Donetsk reg., Shakhtarsk distr. Nadyeina, 2006 NST NST (9) KW33190 Kyrovograd reg., Holovanivka distr. NST, L NST (10) Kondratyuk, 1983 KW27645 Mykolaiv reg., Bratsk distr. Makarevych, 1950 NST, L NST (6), L (6), PL (1) KW33618 Dnipropetrovsk reg. and distr. Hajova, 1960 NST, L, PL NST (2), L (5), PL (1) KW33628 Volyn reg., Kamin-Kashirsk distr. Bradis, 1940 L, PL NST (1), L (2), PL (1) KW62763 Ivano-Frankivsk reg., Verkhovina distr. Blum, 1978 L, PL NST (1), L (2), PL (1)

* NST – norstictic acid, L – lichesterinic acid, PL – protolichesterinic acid; ** content of substances indicated in brackets are: 1 – traces, 2–3 – minor, 4–5 – medium, 6–10 – major. examine the effects of environmental, geographical, follows Prydatko (1998; http://biomodel.info/training- chemical and morphological variables on the quantita- package/ukraine-nature-agricultural-zoning), and is shown tive morphometric thallus parameters (such as width of in Fig. 1E. the main and terminal branches, and size of the thalli), we performed an analysis of variance (ANOVA). Nominal variables included in ANOVA were: occurrence in one Results of the 13 populations and five natural zones of Ukraine, in three specific habitats, at four substrata and three Morphology classes of altitude above sea level (Table 2), the presence of norstictic acid detected by TLC and presence of We found a strong correlation between the isidioid projections (Table 3). Only values with high size of thalli and the presence of isidioid pro- support are shown in Table 5 and discussed. The varia- jections (Fig. 2, Table 5). Thalli without bles included in the PCA were: width of the main isidioid projections were smaller than thalli branch, width of the terminal branch and size of the thalli. The resulting PCA biplots were selected accord- with this trait. However, we found no clear ing to the maximum variance of projection along each regional differentiation in the frequency of component (Fig. 3). isidioid projections. The percentage of speci- To analyze the pairwise correlation between the pres- mens with isidioid projections varied greatly ence and absence of norstictic acid and the geographical distance between C. aculeata specimens, we performed a among populations (0–100% of specimens Mantel test with 99 permutations, as implemented in per population, Table 3). While every speci- Genalex (Peakall & Smouse 2006; Fig. 4). In total, 256 men investigated in the populations of An- specimens of the C. aculeata s. lat. analyzed by TLC tratsit and Chatyr Dag had isidioid projec- (Table 2) were included in this study. tions, none of the specimens from Arabat The distribution map for the C. aculeata s. lat. speci- mens studied was drawn in ArcGis.10 (http://www.esri. Split Botanical Reserve had this characteris- com/software/arcgis). The natural zonation of Ukraine tic (Table 3). 848 THE LICHENOLOGIST Vol. 45

4.6 4.6

4.4 4.4 4.2 4.2 4.0

3.8 4.0 . 3 6 3.8 3.4 Size of the thalli (cm) Size of the thalli (cm) 3.6 3.2 3.4 3.0 AB 2.8 3.2 Isidia– Isidia+ NST– NST+ . 5 0 0.52 4.8 . 4.6 0 51 4.4 0.50 4.2 4.0 0.49 3.8 . 3.6 0 48 . 3 4 0.47 3.2 Size of the thalli (cm) 3.0 0.46 2.8 0.45 2.6 Width of the terminal branches (cm) CD . 2 4 0.44 Sand Soil Granite Soil on NST– NST+ with mosses limestones

Mean; Mean ±SE; Mean ±0.95 % confidence interval

Fig. 2. Relationships among the 122 specimens of Cetraria aculeata group between thallus size and presence or absence of isidioid projections (A, t ¼ -- 3Á751, P ¼ 0Á0003, df ¼ 120), presence or absence of norstictic acid (B, t ¼ 1Á996, P ¼ 0Á048, df ¼ 74) and substratum groups (C, t ¼ 2Á222, P ¼ 0Á029, df ¼ 76), and between width of terminal branches and presence or absence of norstictic acid (D, t ¼ -- 2Á146, P ¼ 0Á033, df ¼ 120).

Our systematically sampled dataset revealed ever, an ANOVA applied to the morpho- rather small differences in morphometric metric variables of C. aculeata s. lat. in relation traits, that are essential for species identifica- to regional effects revealed significant dif- tion, such as the width of the main and termi- ferences between populations (Table 5). nal branches or the size of the thalli (Table 3). Continuous variation within the three mor- The CV of these characteristics showed little phometric characteristics analyzed was also or only average variation in the entire data- confirmed by PCA (Fig. 3). The first two set, as well as within each population. How- components (axes) of PCA explained 77Á8% 2013 Cetraria steppae is conspecific with C. aculeata—Nadyeina et al. 849

Table 5. Results of an analysis of variance testing the effect of environmental, geographical and morphological variables on the morphometric parameters of Cetraria aculeata s. lat. individuals representing different parts of Ukraine (only significant values are shown, n ¼ 122).

Morphometric parameters Source of variation Sum of Squares df FP

Width of main branch Population 1Á22 4 4Á00 0Á0046 Substratum 0Á47 1 6Á22 0Á0142 Width of terminal branch Population 0Á06 4 3Á54 0Á0094 Size of the thalli Population 41Á31 4 10Á21 <0Á0001 Isidia 6Á16 1 6Á10 0Á0152

of variance (the two axes explained 43Á4% minor amounts of nephrosterinic and iso- and 34Á4% of the variance). nephrosterinic acids have been detected by HPLC (Thell & Ka¨rnefelt 2011). Chemistry The analyzed specimens of C. aculeata s. lat. showed no reactions with spot and micro- The analysis of the specimens by TLC crystal tests. As a control, we tested spot reac- showed that the presence of norstictic acid tions and conducted microcrystal tests with K among the populations varied considerably and Pd to detect norstictic acid in other lichen (Table 3). Specimens distributed in the species: Aspicilia cinerea (L.) Ko¨rb., Cladonia Forest zone of Ukraine and in the Carpa- symphycarpia (Flo¨rke) Fr. and Pleurosticta thians always lacked norstictic acid (Table acetabulum (Neck.) Elix & Lumbsch. These 3, Fig. 1E). Additionally, we detected liches- species developed red spots with K or Pd terinic and/or protolichesterinic acids in the and displayed typical reddish crystals in the medulla of some of the specimens, as have microcrystal tests. Therefore, we conclude others in the literature (Ka¨rnefelt 1986; that, in the case of Cetraria aculeata s. lat., Vainshtein et al. 1990). In contrast to the spot reactions and the microcrystal test cannot northern populations of C. aculeata s. lat., be used to detect norstictic acid. some of the specimens from the southern populations in the Steppe zone and the Relationship between morphology, Crimean Mountains contained norstictic chemistry, geographical and acid (17–100% of specimens per population; environmental factors Table 3). HPLC revealed a very broad range of nor- We found a significant correlation between stictic acid content in each specimen (Table thallus size and the presence of norstictic 4). The specimens collected from the same acid, as detected by TLC (Fig. 2). Samples localities contained very different quantities, containing norstictic acid were slightly smaller and some had only traces which could not than samples where norstictic acid could not be detected by TLC. Surprisingly, traces of be detected by TLC. At the same time, speci- norstictic acid were detected by HPLC even mens lacking norstictic acid had narrower in specimens from northern Ukraine and the terminal branches than specimens with nor- Carpathians, where no norstictic acid was stictic acid (Fig. 2). Specimens growing on detected by TLC (Table 4). With HPLC we bare soil and sand had smaller thalli than also detected traces of fumarprotocetraric specimens collected on granite or limestone and connorstictic acids in several specimens, outcrops (Fig. 2). which have never been reported before for All three morphometric thallus variables C. aculeata s. lat. Rangiformic acid was differed between the populations of C. mentioned once for C. aculeata from New aculeata s. lat. The width of the main thallus Zealand (Galloway 1985), and occasionally branch also depended on the substratum, 850 THE LICHENOLOGIST Vol. 45

3 NST– Isidia– NST+ Isidia+ 2

1

0

–1

–2

–3 A B

Population 3 granite A 1 limestone B 2 2 sand C 3 D 4 soil E 5 1 F 6 F 7 0 H 8 I 9 J 10 –1 K 11 L 12 –2 M 13

–3 CD

–3–2–10123 –3–2–10123

Fig. 3. PCA results based on the morphometric characteristics of the C. aculeata s. lat. in Ukraine, n ¼ 122. A, specimens differing chemically are shown; B, specimens with and without isidioid projections are marked; C, speci- mens preferring different substrata are distinguished; D, specimens belonging to different populations are indicated. and the presence of isidioid projections was The overall linear fit of the Mantel test for correlated with thallus size (Table 5). No geographical distance and chemical identity correlations were found between morpho- was 7Á015–7 (P < 0Á001, Fig. 4B), but only metric features and the five natural zones 5Á385–4 (P ¼ 0Á6, Fig. 4A) for short distan- of Ukraine, the three specific habitats, or ces up to 50 m. the three classes of altitude above sea level (Table 5). A continuous scatter plot from a PCA of Discussion the morphometric features of the C. aculeata Taxonomic status s. lat. specimens (Fig. 3) had no structure when compared to the presence of isidioid Our analyses of the morphological, chemical, projections, norstictic acid content, substrata ecological and geographical characteristics preference and sampling in different popula- of C. aculeata s. lat. specimens, originating tions. from 13 populations (Tables 2 & 3), showed We found a weak positive correlation be- a continuous variation in morphometric fea- tween the presence of norstictic acid and a tures, as well as in the presence of isidioid large geographical distance up to 800 km. projections and norstictic acid. Differences 2013 Cetraria steppae is conspecific with C. aculeata—Nadyeina et al. 851

1 1 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 Norstictic acid: 0 - absent, 1 - present 0 - absent, Norstictic acid: 0 10 20 30 40 50 0 200 400 600 800 ABDistance (m) Distance (km)

Fig. 4. Linear fit of Mantel test of geographical distance and chemical identity (absence or presence of norstictic acid) among pairs of specimens, n ¼ 256. A, small distances up to 50 m, parameters estimated with a linear fit of 5Á385–4, P < 0Á6; B, large distances up to 800 km, parameters estimated with a linear fit of 7Á015–7, P < 0Á001.

between populations in the morphometric heavy metal ions into oxalates is well-known, traits examined (Tables 3 & 5) explained and seems to be a way of coping with the local infraspecific variability and did not sup- effects of toxic elements (Jones & Wilson port subdivisions among the specimens of 1985; Purvis 1996). Norstictic acid is also Cetraria aculeata s. lat. we studied (Fig. 3D). known to form complexes with other toxic Seven out of 13 populations were homo- metals, including copper (Purvis et al. 1987). geneous in norstictic acid content when We found no correlation between the pres- analyzed by TLC, while the other six popula- ence of norstictic acid and any of the envi- tions contained chemically distinct indivi- ronmental factors analyzed (Table 5), even duals (Table 3). We found a general trend though we expected specimens with nor- for specimens lacking norstictic acid to have stictic acid (revealed by TLC) to prefer geo- larger thalli and narrower terminal branches graphically different regions (according to (Fig. 2B & D). Therefore the presence of Ka¨rnefelt 1986) or to occur mainly on carbo- norstictic acid depends on the size of the naceous substrata (according to Hauck et al. thallus, which reflects the ontogenetic stage, 2010). The distribution pattern of norstictic and thus should not be considered for taxo- acid in C. aculeata s. lat. did not correlate nomic purposes. An analogous trend has with the natural zones of Ukraine, habitats, been found for perlatolic and usnic acids in substrata or different altitudinal levels. How- another terricolous lichen species, Cladonia ever, specimens with norstictic acid (TLC) stellaris (Opiz) Pouzar & Veˇzda (Fahselt were limited to southern regions in Ukraine 1984). We hypothesize that norstictic acid (the forest-steppe, steppe zones, and the biosynthesis is active during the early stages Crimean Mountains), where they co-occur of thallus development, appearing shortly with specimens lacking norstictic acid. In after thallus fragmentation, and declines or contrast, specimens without norstictic acid becomes deactivated later, once the thalli (TLC) grow throughout all of Ukraine (Table have reached a certain size. We may also 3, Fig. 1E). The sporadic presence of speci- speculate that norstictic acid plays an im- mens with norstictic acid in southern, but portant role in the complexation with heavy not northern, areas supports the hypothesis metals occurring in the substratum, which that norstictic acid plays an adaptive role in would otherwise be toxic for the small thalli. lichens growing in an arid climate (Hauck et The ability of lichen thalli to incorporate al. 2010). A similar trend has been identified 852 THE LICHENOLOGIST Vol. 45 in the terricolous lichen Flavocetraria nivalis Mendoza et al. 2011; Ferna´ndez-Mendoza & (L.) Ka¨rnefelt & A. Thell, which has higher Printzen 2013; Printzen et al. 2013)1 re- concentrations of usnic acid in colder en- vealed pleistocene diversification within the vironments (Bjerke et al. 2004), and for the C. aculeata group into several genetic clusters. epiphytic Pseudevernia furfuracea (L.) Zopf, The only small population from Ukraine which contains olivetoric or physodic acids, (Crimean Mountains) of five individuals with depending on climate (Martellos 2003). These norstictic acid was included in the last two support Hale’s theory (Hale 1956) that the studies, and it appeared to be related to the synthesis of lichen secondary compounds is Mediterranean phylogenetic clade. However, governed by climatic gradients. Neverthe- more extensive sampling of C. aculeata s. lat. less, several other studies have found no evi- from Ukraine, based on a subset of the spe- dence for a climatic dependency of lichens cimens used in our study, revealed shared secondary chemistry (Halvorsen & Bendiksen haplotypes with other genetic clusters, but 1982; Fahselt 1984). no relationship between the genetic struc- Mantel tests revealed a significant spatial tures of populations with norstictic acid pro- autocorrelation between the norstictic acid duction (Lutsak et al. 2012). content (as detected by TLC) and the geo- Further evidence for the incomplete speci- graphical distance between the individuals ation within C. aculeata s. lat. comes from the sampled (Fig. 4B). This reflects regional dif- gene flow between different phylogenetic ferences in norstictic acid content (as mea- clusters within C. aculeata (Ferna´ndez-Men- sured by HPLC) with only traces of norstic- doza & Printzen 2013), and shared haplo- tic acid (not detectable by TLC) in northern types among different operational taxonomic Ukraine and partly higher concentrations in units, such as C. steppae, C. crespoae and C. the southern regions. However, we found no aculeata s. str. represented from geographi- autocorrelation at local scales (Fig. 4A). cally distinct areas (Printzen et al. 2013). This indicates that local habitat characteris- The speciation seems to be very slow for tics do not strongly influence the presence taxa with vegetative reproduction only, where or absence of norstictic acid in C. aculeata s. natural selection is determined by random lat. Pe´rez-Ortega et al. (2012) studied ex- mutations but not recombinations. Cetraria treme cases of phenotypic plasticity in C. aculeata s. lat. includes several genetically aculeata s. lat., including both distinctly va- distinct clades related to the phylogeographi- grant specimens with thick thalli and ‘typical’ cal history of the species (Ferna´ndez-Men- C. aculeata from the same habitats in Spain. doza & Printzen 2013; Printzen et al. 2013), These thick vagrant specimens did not con- but these do not correspond to secondary tain norstictic acid and thus, according to chemistry or morphological characteristics. Ka¨rnefelt (1986), were classified as C. acu- Higher levels of norstictic acid production in leata s. lat. (Pe´rez-Ortega et al. 2012). The C. aculeata s. lat. in southern Ukraine may be study of Pe´rez-Ortega et al. (2012) revealed connected to the aridity of glacial permafrost anatomical and physiological differences be- (Hewitt 1999). Acid production seems to be tween the thick vagrant and ‘typical’ individ- selectively activated depending on the micro- uals of C. aculeata s. lat. from the same habi- climate and the developmental stage of thalli. tats. The authors found, however, shared Possible reasons for the chemical variation haplotypes between those morphs and thus in C. aculeata s. lat. populations may be the emphasized that the vagrant morphs are large-scale geographical and climatic depen- adaptations to aridity. We consider this as dence of gene expression, photobiont switch- further evidence for the morpho/chemical ing and microbial inhabitants (Muggia et al. variability within populations of C. aculeata s. lat. being due to ecologtical (microclimate) and not taxonomic reasons. 1All representatives of C. aculeata group were pooled The recent worldwide multigene studies of together and treated as ‘‘C. aculeata s. lat.’’ in Printzen Cetraria aculeata s. lat. populations (Ferna´ndez- et al. (2013), but not in other studies. 2013 Cetraria steppae is conspecific with C. aculeata—Nadyeina et al. 853

2009; Bates et al. 2011; Ferna´ndez-Mendoza steppae (Blum 1996; Blum et al. 2009). In- et al. 2011; Hodkinson et al. 2012). In a cluding C. aculeata s. lat. in the Red Data recent study, Printzen et al. (2012) demon- Book will highlight the importance of con- strated that the composition of alphapro- serving sand dunes in the Forest natural teobacterial communities in geographically zone of Ukraine, as well as rock outcrops in distant populations of C. aculeata is affected the Crimean and Carpathian Mountains, by environmental factors, but no relationship where the species is even rarer than in the between these communities and secondary steppes. Cetraria aculeata already has an metabolite production has so far been found. approved national conservation status in Our results support a decision to merge C. neighbouring areas (Lo ko¨s & To´th 1996; steppae Savicz with C. aculeata (Schreb.) Fr. Moutchnik & Zavarzin 2005; Lisˇka et al. and to treat both names as synonyms. The 2008). Moreover, the other member of C. features currently used for the delimitation aculeata s. lat., which is difficult to designate at species level are not appropriate because in the field, is C. muricata (Printzen et al. the differences in the morphological features 2013), a species that is also of conservation are too small, the presence and quantity of concern in neighbouring countries (Golubkov norstictic acid varies greatly, and the sub- & Kobzar 2005; Lisˇka et al. 2008). strata and habitats occupied by lichens are To assess the national threat category of very diverse. Cetraria steppae Savicz may be C. aculeata s. lat., we took into consideration regarded as a chemotype only, reflecting an that the area of occupancy by this taxon in infraspecific variation, even though norstictic Ukraine has been constant during recent acid has been detected in the type material of decades. Just two localities have disappeared C. steppae by both TLC and HPLC (Table due to habitat destruction in the Odessa and 4). It is commonly accepted that variation Kharkiv regions during the last 100 years in the concentrations of particular secondary (according to herbaria data from KW and lichen substances does not provide sufficient KHER), which corresponds to 2Á4% of the grounds for taxonomic differentiation (Hawks- total number of localities known for the worth 1976; Elix & Stocker-Wo¨rgo¨tter 2008). taxon. However, the species inhabit vulnera- As a consequence, we recommend formally ble biotopes and recovery after disturbance is synonymizing C. steppae Savicz with C. acu- expected to be very slow because of vagrancy leata (Schreb.) Fr.: and the species’ spatially limited vegetative reproduction (Heinken 1999). We assessed the total national area of oc- Cetraria aculeata (Schreb.) Fr. cupancy of C. aculeata s. lat. as nearly 0Á136 2 Nov. Sched. Critic. Lich. 4: 32 (1826).—Lichen aculeatus km by adding: 1) the area of occupancy Schreb., Spicil. Flor. Lipsiens. 125 (1771). of 13 local populations (each covered very Cetraria steppae (Savicz) Ka¨rnefelt, Bryologist 96: 400 small patches of up to 1000 m2) equal to (1993).—Coelocaulon steppae (Savicz) Barreno & Va´zquez, 2 Lazaroa 3: 329 (1982).—Cornicularia steppae Savicz, Notul. 0Á013 km , and 2) the area of occupancy of Syst. Inst. Cryptog. Horti bot. Petropol. 3: 187 (1924); type: the other 123 localities known in Ukraine, Ukraine, Askania-Nova, Kherson reg., 1924, Oxner (LE, as documented in herbaria (KW, KHER), lectotype!). which is equal to at least 0Á123 km2. Thus, Coelocaulon stepposa (Mereschk.) S. Y. Kondr., Flora Lyshajnykiv Ukraini 2(2): 296 (1993).—Cetraria tenuis- the total national area of occupancy of C. sima f. stepposa Mereschk., Annals and Magaz. Nat. His- aculeata s. lat. is considerably less than the 2 tory 9(8): 249 (1921); type: Rossia meridio-orientalis, 20 km recommended for the ‘vulnerable’ Tauria, Mereschkowsky (KAZ). threat category (VU) according to criterion D2 of IUCN (IUCN Standards and Petitions Conservation status: Ukrainian case Subcommittee 2011). Finally, the threat category of C. aculeata s. lat. in Ukraine is We suggest including Cetraria aculeata s. assessed as vulnerable (VU). This corresponds lat. in the next edition of the Red Data Book with the status of C. steppae in the current of Ukraine, which will replace the entry of C. edition of the Red Data Book of Ukraine 854 THE LICHENOLOGIST Vol. 45

(Blum et al. 2009) and Russia (Golubkova et Ferna´ndez-Mendoza, F., Domaschke, S., Garcı´a, M. al. 2008). A., Jordan, P., Martı´n, M. P. & Printzen, C. (2011) Population structure of mycobionts and photo- We thank Prof. O.Ye. Khodosovtsev (Kherson) for bionts of the widespread lichen Cetraria aculeata. valuable advice and for sending herbarium materials Molecular Ecology 20: 1208–1232. (KHER), as well as Prof. S.Ya. Kondratyuk (Kyiv), Galloway, D. J. (1985) Flora of New Zealand. Lichens. Prof. C. Printzen (Frankfurt on Main) and three anony- Wellington, New Zealand: Hasselberg. mous referees for their useful comments. We are grateful Golubkov, V. V. & Kobzar, N. N. (2005) Lichens. In to Prof. M. P. Andreev (Saint-Petersburg) for sending Red Data Book of the Republic of Belarus: Plants (L. the type specimen (LE220); Prof. V.P. Heluta and O.V. I. Khoruzhik, ed.): 355–383. Minsk: Belarusskaia Vasyluk (Kyiv) for sampling the Ratno and Antratsit Entsiklopedia imeni Petrusia Brovka [In Russian]. populations; Dr. F. Ferna´ndez-Mendoza (Frankfurt on Golubkova, N. S., Dombrovskaia, A. V., Istomina, N. Main) for help with the sampling design for population B., Makarova, I. I., Makryi, T. V., Muchnik, E. study; Dr. L. Lo ko¨s (Budapest), Dr. A. Senkandes¸ler Ye., Pchelkin, A. V., Sedelnikova, N. V., Skirina, I. (Izmir), Dr. A. Tsurikov (Homel), and Dr. A. Yatsina F., Tolpisheva, T. U. et al. (2008) Lichens. In Red (Minsk) for providing several references; and Dr. S. Book of the Russian Federation: Plants and Fungi Dingwall (Birmensdorf ) for revising the English of the (Y. P. Trutnev, ed.): 703–752. Moskwa: Tovari- manuscript. We thank the Rufford SGF for partial fund- shestvo nauchnyh izdanij KMK [In Russian]. ing of this study, as well as the Swiss State Secretariat for Gupta,V. K., Darokar, M. P., Shanker, K., Negi, A., Education and Research (SER). 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